Process for separation and isolation of AHF and fibronectin from blood plasma

ABSTRACT

A process for the separation and isolation of AHF and fibronectin from blood plasma by adding a sulfated mucopolysaccharide to blood plasma to a concentration of 0.15-0.25 mg/ml of plasma, cooling the plasma to 0° C.-15° C. centrifuging the plasma to isolate a precipitate which forms and then removing the AHF-rich supernatant liquid from the precipitate. 
     The precipitate is washed in a slightly basic solution of dilute salts and then dissolved in a buffered solution of chaotrophic salts of the Hofmeister series having an ionic strength from 0.01-1. Fibronectin is then separated, by chromotographic means, from the dissolved precipitate, which contains fibronectin and fibrinogen. 
     In a preferred embodiment, 0.20 mg of sodium heparin per milliliter of human plasma is employed with the heparinized plasma solution cooled for 3 hours at a temperature of 2° C.-4° C.

BACKGROUND OF THE INVENTION

This invention is concerned with a method for obtaining significantamounts of certain components of blood plasma.

The important components of blood plasma with which the process of thisinvention is primarily concerned are the antihemophilia factor (AHF) andplasma fibronectin [cold-insoluble globulin - CIg]. Each of thecomponents obtained according to the process of this invention isextremely valuable in therapeutic applications. The AHF is known, ofcourse, as the single significantly effective agent for use in treatinghemophilia A.

It has been demonstrated in recent years that the fibronectin (CIg)component of human plasma is especially useful in speeding tissuerecovery in trauma, particularly burns. The affinity of fibronectin tocollagen and/or fibrin may be of significance in the mode of its action.

The infusion of plasma fractions enriched with fibronectin has beeneffective in reducing general septsis from severe trauma and burns. Ithas been found that a reduction in the body's level of fibronectinoccurs following trauma, and that the sooner fibronectin is restored tonormal levels, the faster recovery takes place; presumably related to anincrease in the removal of damaged tissues, reduction of infection, andgeneral wound healing.

Of concern to the growing application of fibronectin therapy is thedepletion of plasma available for the isolation of AHF. This has vastlyincreased the need for an effective and efficient large scale processfor separating plasma fractions of predominating fibronectin from thefractions of predominantly AHF activity.

Past methods have suffered from difficulties in effectively separatingthe desired constituents and isolating them on a large scale.

The present invention utilizes a number of individually known steps incombination with new approaches to develop a new overall process for thelarge scale isolation and purification of fibronectin (CIg) and AHF fromblood plasma.

In the past, the separation of these components has been carried out ononly small scale levels of a milliliter of plasma up to one or twohundred milliliters of plasma. No satisfactory method has previouslybeen available for the processing of significant amounts of plasma suchas over 0.5 liters and even up to 1-5 liters.

It is well know that many unpredicted difficulties are usuallyencountered in attempts to scale up laboratory experimental procedureswith plasma to even minimal commercial levels.

In the past, the attempted recovery of the fibronectin from thecryoprecipitate utilized phosphate and/or Tris (hydroxmethyl)aminomethane type of buffer salts and resulted in low percentagerecoveries of fibronectin.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a process for theseparation of the fibronectin-rich (CIg) fraction and the AHF-richfraction of blood plasma by cryoprecipitation in the presence of a veryspecific concentration of certain sulfated mucopolysaccharides such asheparin, alginic acid sulfate, chitin sulfate, polymannuronic acidsulfate and chondroitin sulfate and the subsequent purification of thefibronectin fraction by chromatographic separations utilizing a uniquebuffer system.

According to the process of the present invention, very significantlygreater percentages of both the fibronectin and AHF components arerecovered compared to existing methods. In addition, the process of thepresent invention, while applicable for small scale plasma separationsto which prior methods have generally been limited, is unexpectedlyuseful in separating the AHF and fibronectin fractions on a large scale,i.e., more than 0.5 liters of blood plasma, with sums of 1-2 litersbeing accomplished with ease. Most significantly, the extremely highpercentage recovery of AHF and fibronectin is applicable even when usingthe process with the larger volumes of blood plasma.

Heparin is the preferred polysaccharide to use and the recommendedconcentration is from 0.15 to 0.25 mg/ml of plasma with the preferredconcentration being about 0.20 mg/ml.

The heparin is added to the plasma, which is then cooled to 0° C.-15° C.and preferably 2°-4° C. for at least one hour, preferably for at leastthree hours.

A fine granular precipitate forms and is collected by centrifugation. Aspeed of 3,000-10,000 xg for 15-20 minutes is generally convenient andefficient.

Approximately, 80-90% of the fibronectin component of the plasma isprecipitated in this manner.

The supernatant liquid, which is rich in AHF, may be freeze-dried orfrozen to prevent loss of activity prior to use or further processing.

The precipitate of the fibronectin fraction is washed gently with anice-cold wash of several dilute salts such as NaH₂ PO₄ and NaCl at aslightly basic pH and recentrifuged for ten minutes at 2°-4° C.,preferably three times.

After washing, there is gently added to the precipitate a buffersolution of a chaotrophic salt of the Hofmeister series having an ionicstrength between 0.01-1, preferably 0.05-0.5 with 0.2 ionic strength asrepresentative of the most used level. Chaotrophic salts such as KSCNand KI buffered in the pH range of 6-8 are preferred, with a solution of0.2 M KSCN, 0.1 M Tris-Cl at pH 7.5 as most often used to dissolve theprecipitate. The buffer is generally warmed to about 37° C.

The precipitate mixture is then warmed for 1-2 hours at about 37° C. Itis important not to disturb the precipitate at this point to avoidirreversible aggregation of the fibronectin-fibrinogen-heparin complex.

After the warming period, the precipitate is partially dissolved. Thenadditional buffer solution is added to dilute to a final concentrationof 0.05 M KSCN, bringing the protein concentration to 2-4 mg/ml.

The dissolved fibronectin and fibrinogen protein complex is thenseparated chromatographically. The solution is applied to a DEAEcellulose (Whatman DE-23) column.

The fibrinogen is usually eluted with 0.05 M KSCN, 0.1 M Tris-Cl at a pHof about 6-8, preferably about 7.5. When absorbance returns to abaseline level, a linear gradient of 0.05 M KSCN, 0.1 M Tris-Cl at pH7.5 (75-300 ml/chamber) is applied, and finally a wash of 0.5 M KSCN,0.1 M Tris-Cl at a pH of about 7.5. Heparin separates from thefibronectin at the end of the gradient.

Also, the fibronectin can be eluted with a single buffer after thefibrinogen has been removed. Any buffer of at least 0.25 ionic strengthand a pH range of 6-8 may be employed, such as 0.25 M NaH₂ PO₄ -Tris pH7.0 or 0.3 M KSCN, 0.1 M Tris-Cl pH 7.5 and preferably 0.25 MPhosphate-Tris pH 7.0 or 0.3 M KSCN, 0.1 M Tris-Cl pH 7.5 to remove thefibronectin from the resin with a minimal amount of contaminatingheparin. However, there is a 10-20% decrease in recovery of fibronectinat this phase. Approximately 45-50% recovery of fibronectin is usuallyobtained with either system.

As to the heparin supernatant fraction, each liter is mixed with ananion exchange resin, for example, 20-30 grams of DEAE cellulose withmicro-crystalline binder (such as Heparasorb, General Diagnostics) for10-15 minutes. The resin is then removed by constant flow centrifugationor macrofiltration.

The supernatant liquid recovered usually has 90-95% of the originalprocoagulant activity. The removal of heparin is confirmed by a thrombintime determination.

In this specification and the claims, it should be understood that theterm blood plasma includes blood plasma itself and cryoprecipitate whichhas been dissolved in dilute salt buffers.

The following representative embodiments will further illustrate thenature of the invention.

ILLUSTRATIVE EMBODIMENTS EXAMPLE 1

1.5 Liters of human blood plasma are warmed at 37° C. for 30 minutes.1.6 mls Of a 200 mg/ml sodium heparin solution (sigma 167 μ/mg) is mixedwith the plasma and the plasma then cooled at 4° C. for 18 hours(overnight) and a precipitate formed. The precipitate is collected bycentrifugation at 4° C. for 30 min. at 9,500 xg. The supernatant liquidis saved.

The precipitate is washed with 1 liter of 0.05 M NaH₂ PO₄, 0.1 M NaCl pH7.2 at 4° C.; centrifuged for 30 minutes at 9,500 xg and the washdiscarded. The precipitate is dissolved in 120 mls of 0.2 M KSCN, 0.1 MTris-Cl at pH 7.5. The total protein content is 484 with 4.03 mg/mlconcentration. The dissolved precipitate is diluted with 0.1 M Tris-ClpH 7.5 to a final concentration of 0.05 M KSCN.

The diluted sample is applied to a 2.5×40 cm DEAE cellulose columnpre-equilibrated with 0.05 M KSCN, 0.1 M Tris-Cl pH 7.5 and the columnwashed with 0.05 M KSCN buffer until the absorbance returned tobaseline. A stepwise buffer, 0.25 M Tris-PO₄, pH 7.0 is used rather thana gradient to elute the fibronectin. A total of 176.5 mg of fibronectinis eluted which represents about 40-45% recovery of total fibronectin,assuming a 250-300 mg/liter starting concentration.

EXAMPLE 2

300 mls Of human blood plasma are warmed to 37° C. To the warm plasma,NaN₃ os added to a dilution of 0.02%, and Trasylol to a dilution of 2mg/ml. To this solution is added sodium heparin to obtain a finalconcentration of 0.2 mg/ml of heparin. The solution is stirred until thematerials are completely dissolved. The solution is cooled in a waterbath at 2° C. for three hours and then centrifuged at 10,000 xg for 30minutes at 2° C. The supernatant liquid contains 90-95% of the AHFmaterial.

The precipitate is washed with a cold solution of 0.05 M NaH₂ PO₄,0.1 MNaCl, at a pH 7.2. The solution is recentrifuged and washed twice again.The precipitate is then dissolved in 0.05 M KI, 0.1 M Tris at a pH of7.5 with 0.02% NaN₃, and 2μ/ml Trasylol to a concentration of 5 mg/ml.

The dissolved precipitate is applied to a 1.5×30 cm DEAE cellulosecolumn and the fibrinogen is eluted with a 0.05 M KI, 0.1 M Tris-Cl pH7.5 buffer. The fibronectin (CIg) is then eluted with a linear gradientof 0.05 M KI, 0.1 M Tris-Cl pH 7.5 buffer. The fibronectin peak waspooled into three fractions adding up to 83 mg from a starting amount of128 mg, yielding a 65% recovery.

EXAMPLE 3

The procedure of Example 1 is repeated using 0.15 mg/ml of heparin withsatisfactory results although the percentage of fibronectin isolated issomewhat less.

EXAMPLE 4

The procedure of Example 1 is repeated but using 0.25 mg/ml of sodiumheparin.

While the invention has been explained by a detailed description ofcertain specific embodiments, it is understood that variousmodifications and substitutions can be made in any of them within thescope of the appended claims which are intended also to includeequivalents of such embodiments.

What is claimed is:
 1. A process for the separation and isolation of AHFand fibronectin fractions from blood plasma comprising:(a) adding asulfated mucopolysaccharide to blood plasma to a concentration of0.15-0.25 mg/ml of plasma; (b) then cooling said plasma to 0° C.-15° C.;(c) centrifuging the plasma to isolate a precipitate which forms; (d)removing the supernatant liquid from the precipitate, said supernatantliquid being rich in AHF; (e) washing the precipitate in a slightlybasic solution of dilute salts; (f) thereafter dissolving saidprecipitate in a buffered solution of chaotrophic salts of theHofmeister series having an ionic strength from 0.01-1; (g) andthereafter separating said fibronectin from the dissolved precipitatewhich contains fibronectin and fibrinogen by chromotographic means.
 2. Aprocess as claimed in claim 1, wherein said mucopolysaccharide isheparin.
 3. A process as claimed in claim 2, wherein said heparin ispresent in an amount of 0.20 mg/ml of plasma.
 4. A process as claimed inany of the preceding claims, wherein said blood plasma is cooled to 2°C.-4° C. for 30 minutes to 3 hours.
 5. A process as claimed in any ofthe preceding claims, wherein said chaotrophic salt has an ionicstrength of from 0.05-0.5.
 6. A process as claimed in any of thepreceding claims, wherein the supernatant liquid is mixed with an anionexchange resin for 10-15 minutes, and thereafter said resin is removed.7. A process for the separation and isolation of AHF and fibronectinfrom blood plasma which comprises adding sodium heparin to human bloodplasma to obtain a concentration of about 0.20 mg of heparin permilliliter of plasma, then cooling the plasma solution to 2° C.-4° C.for 1-3 hours, thereafter centrifuging the precipitate which forms,removing the supernatant liquid which is rich in AHF, removing theheparin from said AHF-rich fraction, washing said precipitate with adilute salt solution at a slightly basic pH, thereafter dissolving saidprecipitate in a buffered solution of chaotrophic salts of theHofmeister series having an ionic strength of about 0.05-0.5, andfinally separating said fibronectin from the fibronectin-fibrinogenmixture by chromotographic means.